Method of pre-treating sample for measuring saccharified amine and method of measuring saccharified amine

ABSTRACT

The present invention provides a method of pretreating a sample containing a glycated amine as an analyte, thereby enabling highly reliable measurement of a glycated amine. A glycated amino acid in the sample is degraded by causing a fructosyl amino acid oxidase (FAOD) to act thereon, and thereafter, a FAOD further is caused to act on the glycated amine as the analyte in the sample to cause a redox reaction. The amount of the glycated amine is determined by measuring the redox reaction. The substrate specificity of the FAOD caused to act on the glycated amino acid may be either the same as or different from that of the FAOD caused to act on the glycated amine. When using the same FAOD, a FAOD is caused to act on the glycated amino acid to degrade it, and thereafter, the sample is treated with a protease to inactivate the FAOD and also to degrade the glycated amine. Then, the same FAOD further is added so that the FAOD acts on the degradation product obtained to cause a redox reaction, and the redox reaction is measured.

TECHNICAL FIELD

[0001] The present invention relates to a method of pretreating a samplefor measurement of a glycated amine and to a method of measuring aglycated amine.

BACKGROUND ART

[0002] Conventionally, the measurement of the amount of an analyte in asample using a redox reaction has been utilized for a wide range ofapplications. For example, such measurement has been utilized formeasuring glycated amines (glycated proteins, glycated peptides,glycated amino acids, etc.) in applications such as biochemicalanalyses, clinical tests, and the like.

[0003] In particular, glycated proteins in blood, especially glycatedhemoglobins in erythrocytes, serve as important indexes in thediagnosis, treatment, etc. of diabetes, because they reflect thepatient's past history of blood glucose levels. Such glycated proteinsin erythrocytes are measured using a redox reaction, for example, in thefollowing manner.

[0004] First, erythrocytes are hemolyzed to prepare a sample. Then, afructosyl amino acid oxidase (hereinafter referred to as “FAOD”) isadded to this hemolyzed sample. The FAOD acts on a glycation site of aglycated protein to cause a redox reaction, thereby forming hydrogenperoxide. The amount of the hydrogen peroxide corresponds to the amountof the glycated protein. Subsequently, a peroxidase (hereinafterreferred to as “POD”) and a reducing agent that develops color byoxidation are added to the sample so that a redox reaction occursbetween the hydrogen peroxide and the reducing agent with the POD as acatalyst. This redox reaction causes the reducing agent to developcolor, and the amount of the hydrogen peroxide can be determined bymeasuring the color developed. As a result, the amount of the glycatedprotein in the erythrocytes can be determined.

DISCLOSURE OF INVENTION

[0005] However, methods of measuring a glycated protein using such aredox reaction have a problem in that, for example, more hydrogenperoxide may be formed than is corresponding to the glycated proteinactually contained in a sample. Besides, depending on the patient, themeasured value of a glycated protein may leap up temporarily, so that areliable measurement cannot be performed. Therefore, further improvementin the accuracy of the measurement is desired in the above-describedmethods. Moreover, not only in the methods using a redox reaction butalso in methods of measuring glycated amine using, for example, anantigen-antibody reaction, improvement in the accuracy of measurement isdesired.

[0006] Therefore, it is an object of the present invention to provide amethod of pretreating a sample for measurement of a glycated amine,thereby enabling highly reliable measurement of the glycated amine.

[0007] In order to achieve the above object, the present inventionprovides a method of pretreating a sample containing a glycated amine asan analyte, including: causing a FAOD to act on a glycated amino acid ora glycated peptide present in the sample other than the glycated amineas the analyte so as to remove the glycated amino acid or the glycatedpeptide by degrading it.

[0008] It is to be noted that “FAOD” merely is a generic name and thesubstrate thereof is not limited to glycated amino acids. For example,FAODs act also on glycated peptides. Furthermore, in the presentinvention, a “glycated peptide” refers to the one with the length thatallows FAODs to act thereon. For example, it refers to the one having 2to 6 amino acid residues. Accordingly, the term “glycated proteins” asused in the present invention includes glycated proteins and alsoglycated peptides with the length that does not allow FAODs to actthereon. Examples of the glycated amine as the analyte include glycatedproteins, glycated peptides, and glycated amino acids.

[0009] The inventors of the present invention have conducted in-depthresearches to improve the accuracy of the measurement and finally foundout the following fact. In whole blood, not only a glycated amine as ananalyte but also a free-state glycated amino acid and a free-stateglycated peptide other than the analyte are present inherently. FAODsalso act on such a glycated amino acid and a glycated peptide. Thus,when a glycated amine is measured using a FAOD as described above, aredox reaction occurs not only between the glycated amine as the analyteand the FAOD, but also between the glycated amino acid or glycatedpeptide other than the analyte and the FAOD, so that the measured valueof the analyte increases seemingly. Moreover, regarding theabove-described problem that some patients show considerable variationin the measured value depending on the time when the blood is collectedeven though the measurement is carried out by the above-described methodwith respect to the whole blood samples collected from the same patientunder the same conditions, the inventors of the present invention alsofound out the following fact. This problem is seen mainly in patientsafter being put on an intravenous drip or the like. For example, if asaccharide such as glucose and any of various amino acids areadministered via an intravenous drip or the like, a glycated amino acidis formed from such exogenous substances. As a result, the glycatedamino acid increases temporarily, which causes the above-describedvariation in measured value. Such a phenomenon is observed not only inthe methods using a redox reaction but also in the methods using anantigen-antibody reaction, for example. Thus, based on these findings,the inventors of the present invention discovered that, even if a wholeblood sample contains a glycation product other than the analyte, suchas a glycated amino acid or glycated peptide present homeostatically oran exogenous glycated amino acid present temporarily, the seemingincrease in the measured value can be suppressed by pretreating thesample in advance so as to cause a degradation FAOD to act on theglycated amino acid or glycated peptide other than the analyte todegrade it as in the present invention. This allows the accuracy of themeasurement to be improved. In addition, since this allows blood to becollected any time regardless of whether or not the patients were put onan intravenous drip, for example, the labor of patients can be reduced.Therefore, by applying the method of pretreating a sample according tothe present invention to, for example, the measurement of a glycatedhemoglobin using the above described methods, the reliability of theglycated hemoglobin as an index in the diagnosis and the like ofdiabetes increases. As a result, the methods become useful in the fieldof clinical medicine and the like.

[0010] Next, the present invention provides a method of measuring anamount of a glycated amine as an analyte in a sample, including:pretreating the sample by the above-described pretreatment method of thepresent invention so as to remove the glycated amino acid or theglycated peptide present in the sample other than the glycated amine asthe analyte by degrading it; then causing a FAOD to act on the glycatedamine to cause a redox reaction; and measuring the redox reaction todetermine the amount of the glycated amine.

[0011] For the reason described above, the accuracy of the measurementcan be improved by such a method for measurement. Hereinafter, a FAODused for degrading the glycated amino acid or the glycated peptide otherthan the analyte is referred to as a “degradation FAOD” and a FAODcaused to act on the glycated amine to measure it is referred to as a“measurement FAOD” in the present invention. Furthermore, the glycatedamino acid or glycated peptide present in the sample other than theglycated amine as the analyte hereinafter also is referred to as the“non-analyte glycation product”.

[0012] Examples of the method for measurement according to the presentinvention include a first method in which FAODs having substratespecificities different from each other are caused to act on anon-analyte glycation product and a glycated amine as an analyte,respectively, and a second method in which the same FAOD is caused toact on them.

[0013] As described later, there are various FAODs, e.g., a FAOD thatacts on a glycated α-amino group, a FAOD that acts on a glycated aminogroup in a side chain (hereinafter also referred to as a “glycatedside-chain amino group) of an amino acid residue such as a lysineresidue or an arginine residue, and a FAOD that acts on both a glycatedα-amino group and a glycated side-chain amino group, and their substratespecificities vary depending on the type of FAODs. In the measurement ofa glycated amine, the amount of the glycated amine can be measured bycausing a FAOD to act on any of the glycated α-amino group, the glycatedside-chain amino group, and both the glycated α-amino group and theglycated side-chain amino group.

[0014] In the first method of the present invention, it is preferablethat the degradation FAOD caused to act on the non-analyte glycationproduct has a substrate specificity different from that of themeasurement FAOD caused to act on the glycated amine as the analyte.With this configuration, the glycation site of the non-analyte glycationproduct is degraded with the degradation FAOD, and then, with regard tothe glycated amine, the glycation site thereof not subjected to theaction of the degradation FAOD is subjected to the action of themeasurement FAOD having a substrate specificity different from that ofthe degradation FAOD. Thus, the influence of the non-analyte glycationproduct can be eliminated so that the accuracy of the measurement isimproved.

[0015] Specifically, when the non-analyte glycation product has aglycated α-amino group and the glycated amine as the analyte has aglycated α-amino group and a glycated side-chain amino group, forexample, it is preferable that the degradation FAOD is specific for aglycated α-amino group, and the measurement FAOD is specific for aglycated α-amino group and a glycated side chain of an amino acidresidue. Since the measurement FAOD acts on both a glycated α-aminogroup and a glycated side-chain amino group, it also acts on thenon-analyte glycation product having a glycated α-amino group when usedin conventional methods, as described above. However, in the presentinvention, since the glycation site of the non-analyte glycation productis degraded with the degradation FAOD specific for a glycated α-aminogroup in advance, there is no chance that the measurement FAOD may actthereon. As a result, the seeming increase in the measured value issuppressed so that the accuracy of the measurement is improved.Moreover, it is possible to cause the measurement FAOD to act only onthe glycated side-chain amino group of the glycated amine since themeasurement FAOD acts on both a glycated α-amino group and a glycatedside-chain amino group as described above and the glycated α-amino groupof the glycated amine also is degraded with the degradation FAOD.Therefore, this method particularly is useful for measurement of aglycated amine that is characterized by the amount of the glycatedside-chain amino group. Examples of such a glycated amine includeglycated lysine having a glycated 1-amino group and glycated albumin.

[0016] When using different FAODs as described above, it is preferablethat the glycated amine is degraded with a protease to give adegradation product of the glycated amine either before or after causingthe degradation FAOD to act on the non-analyte glycation product and theabove-described redox reaction is caused by causing the measurement FAODto act on the degradation product. The degradation of the glycated amineis carried out because, when the analyte is a glycated protein, FAODshave properties that they do not act on glycated proteins easily whereasthey act on the glycated peptides as described above and glycated aminoacids more easily, and act on glycated amino acids still more easilythan on the glycated peptides. Moreover, the reason why the proteasetreatment may be carried out either before or after the degradationtreatment of the non-analyte glycation product is that, since themeasurement FAOD can act also on the glycation site other than that onwhich the degradation FAOD acts as described above, the degradation FAODtreatment does not have any influence on the measurement of the glycatedamine itself. Also, when the analyte is a glycated peptide, it ispreferable to carry out a protease treatment because the FAOD can actstill more easily if the glycated peptide is degraded with a protease tostill shorter glycated peptides or glycated amino acids.

[0017] In the present invention, unless otherwise stated, “a glycatedamino acid and glycated peptide as non-analyte glycation products” referto those contained in a sample before the degradation by the proteasetreatment and do not include a degradation product of a glycated proteinor a glycated peptide as an analyte obtained by the protease treatment.

[0018] Next, as the second method of the present invention, it ispreferable that the degradation FAOD is caused to act on the non-analyteglycation product, thereafter, the glycated amine is degraded with aprotease to give a degradation product of the glycated amine, and theabove-described redox reaction is caused by adding the same FAOD as thedegradation FAOD so that it acts on the degradation product. The secondmethod is useful, for example, when the non-analyte glycation product isa glycated amino acid and the glycated amine as the analyte is aglycated protein or a glycated peptide.

[0019] Specifically, it is preferable that the degradation FAOD isinactivated with the protease. As described above, FAODs have propertiesthat they do not act on glycated proteins easily and act on glycatedamino acids still more easily than on glycated peptides. Thus, when thenon-analyte glycation product is, for example, a glycated amino acid, itcan be said based on chemical kinetics of enzymes that, even though adegradation FAOD is added, it does not act on a glycated protein andhardly acts on a glycated peptide within a treatment period fordegrading the glycated amino acid. However, if the activity of thedegradation FAOD still remains during the protease treatment performedsubsequently, the remaining degradation FAOD acts on a glycated proteindegradation product (such as glycated peptides and glycated amino acids)or a glycated peptide degradation product (such as shorter glycatedpeptides and glycated amino acids) obtained while the glycated amine asthe analyte is being degraded with the protease. Therefore, when themeasurement FAOD is added after the protease treatment, part of theglycated protein degradation product or the like already is subjected tothe action of the degradation FAOD. As a result, contrary to what isintended, the accuracy of the measurement may be deteriorated. However,if the protease treatment performed to degrade a glycated protein or thelike serves to inactivate the remaining degradation FAOD at the sametime as described above, the glycated protein degradation product or thelike obtained by the protease treatment remains unreacted with thedegradation FAOD and thus can react with the measurement FAOD addedsubsequently. As a result, the accuracy of the measurement can beimproved.

[0020] The second method also is useful when the analyte is a glycatedprotein and the non-analyte glycation product is a glycated peptide, forexample. This is because, when the degradation FAOD is caused to act onthe glycated peptide, it does not act on the glycated protein within atreatment period for degrading the glycated peptide.

[0021] On the other hand, even if the degradation FAOD is notinactivated by the protease treatment as described above, highlyaccurate measurement also can be realized by, for example, adjusting theamounts of the degradation FAOD and the measurement FAOD to be added tothe sample. In this case, the ratio (activity ratio A: B) of thedegradation FAOD (A) to the measurement FAOD (B) preferably is set in arange from 1:10 to 1:50,000. When the ratio of the degradation FAOD tothe measurement FAOD is in the above-described range, if the degradationFAOD remains during the protease treatment, the remaining degradationFAOD does not act on the glycated peptide as the analyte, not to mentionon a glycated protein, as easily as on the glycated amino acid as thenon-analyte glycation product, as understood from the chemical kineticsof enzymes. This also applies to the case where the non-analyteglycation product is a glycated peptide and the glycated amine as theanalyte is a glycated protein.

[0022] In the method of the present invention, as the protease, althoughnot particularly limited, at least one protease selected from the groupconsisting of metalloproteinases, bromelain, papain, trypsin, proteinaseK, subtilisin, and aminopeptidase can be used, for example.

[0023] When the analyte is a glycated hemoglobin as described later, theprotease is the one that degrades the glycated hemoglobin selectively,and at least one protease selected from the group consisting ofmetalloproteinases, bromelain, papain, trypsin derived from porcinepancreas, and protease derived from Bacillus subtilis preferably isused. Among these, metalloproteinases and protease derived from Bacillussubtilis are more preferable, and metalloproteinases are particularlypreferable. By using such a protease, only the glycated hemoglobin canbe measured because glycated proteins other than the glycated hemoglobinhardly are degraded with the protease and thus a FAOD hardly acts on theglycated proteins other than the glycated hemoglobin.

[0024] In the present invention, the sample used for measurement is notparticularly limited. For example, the method for measurement accordingto the present invention can be applied to biological samples such aswhole blood, plasma, serum, blood cells, urine, and spinal fluid, drinkssuch as juices, and foods such as soy sauce and Worcestershire sauce.Among these, the method particularly is useful for the blood samplessuch as whole blood, plasma, serum, and blood cells as described aboveand the biological samples other than those, for example.

[0025] Furthermore, even when the whole blood sample contains, forexample, an exogenous glycated amino acid or the like, the measurementstill can be carried out with high accuracy. For example, even thoughthe exogenous glycated amino acid is present in whole blood onlytemporarily, it has a considerable influence on the measured value of aglycated protein or the like when it is contained in whole blood.However, according to the present invention, such an influence can beeliminated.

[0026] While the whole blood sample is not particularly limited, themethod for measurement according to the present invention is useful fora whole blood sample collected from a patient after being put on anintravenous drip, for example. This is because the variation in themeasured value due to an exogenous glycated amino acid being formed isobserved especially in a sample collected from a patient after being puton an intravenous drip.

[0027] In the method for measurement according to the present invention,the analyte is not particularly limited as long as a redox reaction isutilized. For example, the analyte may be components in whole blood,components in erythrocytes, components in plasma, components in serum,components in urine, components in spinal fluid, and the like, and it ispreferably a component in erythrocytes. For example, when a component inerythrocytes is to be measured, whole blood itself may be hemolyzed toprepare a sample, or erythrocytes may be separated from whole blood andhemolyzed to prepare a sample.

[0028] In the method for measurement according to the present invention,examples of the glycated amine as the analyte include glycated proteins,glycated peptides, and glycated amino acids. Specifically, the analytemay be a glycated protein such as a glycated hemoglobin or a glycatedalbuminm. When a glycated hemoglobin as a component in erythrocytes isto be measured, whole blood itself may be hemolyzed to prepare a sample,or erythrocytes are separated from whole blood and hemolyzed to preparea sample, for example.

BRIEF DESCRIPTION OF DRAWINGS

[0029]FIG. 1 is a graph showing the correlation between the amount ofHbA1c measured by the method for measurement using FAODs according toone example of the present invention and that measured using anautomatic analysis apparatus.

BEST MODE FOR CARRYING OUT THE INVENTION

[0030] In the method for measurement according to the present invention,FAODs catalyzing a reaction represented by Formula (1) below preferablyare used. Examples of such FAODs include a FAOD specific for a glycatedamine having a glycated α-amino group (hereinafter referred to as a“FAOD-α”), a FAOD specific for a glycated amine having a glycated aminogroup in a side chain of an amino acid residue (hereinafter referred toas a “FAOD-S”), and a FAOD specific for both a glycated amine having aglycated α-amino group and a glycated amine having a glycated aminogroup in a side of an amino acid residue (hereinafter referred to as a“FAOD-αS”).

R¹—CO —CH₂—NH—R²+H₂O+O₂→R¹—CO—CHO+NH₂—R²+H₂O₂  (1)

[0031] In Formula (1), R¹ denotes a hydroxyl group or a residue derivedfrom the sugar before glycation (i.e., sugar residue). The sugar residue(R¹) is an aldose residue when the sugar before glycation is aldose, andis a ketose residue when the sugar before glycation is ketose. Forexample, when the sugar before glycation is glucose, it takes a fructosestructure after glycation by an Amadori rearrangement. In this case, thesugar residue (R¹) becomes a glucose residue (an aldose residue). Thissugar residue (R¹) can be represented, for example, by

—[CH(OH)]_(n)—CH₂OH

[0032] where n is an integer of 0 to 6.

[0033] In Formula (1), R² is not particularly limited. However, when theglycated amine is a glycated amino acid or a glycated peptide, there isa difference between the case where an α-amino group is glycated and thecase where an amino group other than the α-amino group (i.e., an aminogroup in a side chain of an amino acid residue) is glycated.

[0034] In Formula (1), when an α-amino group is glycated, R² is an aminoacid residue or a peptide residue represented by Formula (2) below. Theabove-described FAOD-α and FAOD-αS specifically catalyze the reactionrepresented by Formula (1) in this case.

—CHR³—CO—R⁴  (2)

[0035] In Formula (2), R³ denotes an amino-acid side chain group. R⁴denotes a hydroxyl group, an amino acid residue, or a peptide residue,and can be represented, for example, by Formula (3) below. In Formula(3), n is an integer of 0 or more, and R³ denotes an amino-acid sidechain group as in the above. When n is an integer of more than 1, theamino-acid side chain groups may be either the same or different.

—(NH—CHR³—CO)_(n)—OH  (3)

[0036] In Formula (1), when an amino group other than the α-amino groupis glycated (i.e., an amino-acid side chain group is glycated), R² canbe represented by Formula (4) below. The above-described FAOD-S andFAOD-αS specifically catalyze the reaction represented by Formula (1) inthis case.

—R⁵ CH(NH—R⁶)—CO—R⁷  (4)

[0037] In Formula (4), R⁵ denotes a portion other than the glycatedamino group in the amino-acid side chain group. For example, when theglycated amino acid is lysine, R⁵ is as follows.

CH₂—CH₂—CH₂—CH₂—

[0038] For another example, when the glycated amino acid is arginine, R⁵is as follows.

—CH₂—CH₂—CH₂—NH—CH(NH₂)—

[0039] In Formula (4), R⁶ denotes hydrogen, an amino acid residue, or apeptide residue, and can be represented, for example, by Formula (5)below. In Formula (5), n denotes an integer of 0 or more, and R³ denotesan amino-acid side chain group as in the above. When n is an integer ofmore than 1, amino-acid side chain groups may be either the same ordifferent.

—(CO—CHR³—NH)_(n)—H  (5)

[0040] In Formula (4), R⁷ denotes a hydroxyl group, an amino acidresidue, or a peptide residue, and can be represented, for example, byFormula (6) below. In Formula (6), n is an integer of 0 or more, and R³denotes an amino-acid side chain group as in the above. When n is aninteger of more than 1, the amino-acid side chain groups may be eitherthe same or different.

—(NH—CHR³—CO)_(n)—OH  (6)

[0041] Examples of the FAOD-α specific for a glycated α-amino groupinclude a commercially available product named Fructosyl-Amino AcidOxidase (FAOX-E) (manufactured by Kikkoman Corporation) and FAODsderived from the genus Penicillium (JP 8(1996)-336386 A). Examples ofthe FAOD-S specific for a glycated side chain of an amino acid residueinclude FAODs derived from the genus Fusarium (“Conversion of SubstrateSpecificity of Amino Acid Oxidase Derived from Fusarium oxysporum” byMaki FUJIWARA et al., Annual Meeting 2000, The Society forBiotechnology, Japan). Furthermore, examples of FAOD-αS specific forboth a glycated α-amino group and a glycated side chain group of anamino acid residue include a commercially available product named FOD(manufactured by Asahi Chemical Industry Co., Ltd.), FAODs derived fromthe genus Gibberella (JP 8(1996)-154672 A), FAODs derived from the genusFusarium (JP 7(1995)-289253 A), and FAODs derived from the genusAspergillus (WO 99/20039).

[0042] Hereinafter, the method for measurement according to the presentinvention will be described in detail with reference to the followingexamples, in which a glycated protein derived from blood cells ismeasured using a whole blood sample containing a glycated amino acid asa non-analyte glycation product. In the present invention, unlessotherwise stated, “a glycated amino acid as a non-analyte glycationproduct” refers to the one contained in the sample before starting themeasurement and does not include a degradation product of the glycatedprotein as the analyte obtained by the treatment with a protease.

[0043] (First Embodiment)

[0044] The present embodiment is one example of the first method, inwhich a FAOD-α is used to degrade the glycated amino acid and a FAOD-αSis used to measure the glycated protein.

[0045] First, whole blood is hemolyzed to prepare a hemolyzed sample.The method of causing the hemolysis is not particularly limited, and canbe, for example, a method using a surfactant, a method using ultrasonicwaves, a method utilizing a difference in osmotic pressure, and a methodusing a freeze-thawing technique. Among these, the method using asurfactant is preferable because of its simplicity in operation, etc.

[0046] As the surfactant, for example, non-ionic surfactants such aspolyoxyethylene-p-t-octylphenyl ether (e.g. Triton series surfactants),polyoxyethylene sorbitan alkyl ester (e.g. Tween series surfactants),polyoxyethylene alkyl ether (e.g. Brij series surfactants), and the likecan be used. Specific examples are products named Triton X-100,Tween-20, Brij 35, and the like. The conditions for the treatment withthe surfactant usually are as follows: when the concentration of bloodcells in the solution to be treated is in the range from 1 to 10 vol %,the surfactant is added so that its concentration in the solution fallsin the range from 0.1 to 1 wt %, and stirred at room temperature forabout 5 seconds to 1 minute.

[0047] Furthermore, when utilizing the difference in osmotic pressure,to the whole blood is added 2 to 100 times its volume of purified waterto cause hemolysis, for example.

[0048] Next, the hemolyzed sample is treated with a protease. Thisprotease treatment is carried out to degrade the glycated protein sothat a FAOD described later can act thereon more easily. The type of theprotease is not particularly limited, and for example, theabove-described proteinase K, subtilisin, trypsin, aminopeptidase,papain, metalloproteinases, and the like can be used. The proteasetreatment usually is carried out in a buffer, and the conditions of thetreatment are determined as appropriate depending on the type of theprotease used, the type and the concentration of the glycated protein asthe analyte, etc.

[0049] When the sample is treated using trypsin as the protease, theprotease treatment is carried out, for example, under the conditions asfollows: the concentration of the protease in the reaction solution inthe range from 100 to 6000 U/l; the concentration of blood cells in thereaction solution in the range from 0.2 to 5 vol %; the reactiontemperature in the range from 20° C. to 50° C.; the reaction period inthe range from 10 minutes to 20 hours; and the pH in the range from 6 to9. The treatment usually is carried out in a buffer. The type of thebuffer is not particularly limited, and for example, Tris-HCl buffer,phosphate buffer, EPPS buffer, PIPES buffer, and the like can be used.

[0050] Next, the hemolyzed sample treated with the protease is treatedwith a FAOD-α catalyzing the reaction represented by Formula (1) above,more specifically the reaction represented by Formula (7) below.

R¹—CO—CH₂—NH—CHR³—COOH+H₂O+O₂→R¹—CO—CHO+NH₂—CHR³—COOH+H₂O₂  (7)

[0051] In Formula (7), R¹ denotes a sugar residue as in the above, andR³ denotes an amino-acid side chain group as in the above.

[0052] By this treatment, the glycated amino acid having a glycatedα-amino group and the glycated α-amino group of the glycated proteindegradation product contained in the hemolyzed sample are degraded.

[0053] According to this FAOD-α treatment, among various glycated aminoacids, the one having a glycated side-chain amino group remains withoutbeing degraded. However, considering the ratio of the glycated aminoacid having a glycated side-chain amino group to the glycated aminoacids as a whole and the ratio of the same to amino acid residues havinga glycated side-chain amino group in glycated proteins, it can be saidthat the influence of the remaining glycated amino acid is small so thatthe accuracy of the measurement can be improved sufficiently.

[0054] The FAOD-α treatment is carried out, for example, under theconditions as follows: the concentration of the FAOD-α in the reactionsolution in the range from 10 to 5000 U/l, the concentration of theblood cells in the reaction solution in the range from 0.5 to 20 vol %,the reaction temperature in the range from 20° C. to 50° C., thereaction period in the range from 1 minute to 1 hour, and the pH in therange from 6 to 9. The FAOD-α treatment usually is carried out in abuffer, and the same buffers as in the protease treatment also can beused in the FAOD-α treatment.

[0055] Subsequently, the hemolyzed sample treated with the FAOD-α istreated further with a FAOD-αS. As described above, the FAOD-αS acts onboth a glycated α-amino group and a glycated side-chain amino group.However, since the glycated protein degradation product has been treatedwith the degradation FAOD-α in advance, it is possible to cause thismeasurement FAOD-αS to act only on the glycated side-chain amino groupof the glycated protein degradation product.

[0056] Similarly to the above-described protease treatment, this FAOD-αStreatment preferably is carried out in a buffer. The type of the bufferis not particularly limited, and the same buffers as in the proteasetreatment also can be used in the FAOD-αS treatment.

[0057] The FAOD-αS treatment is carried out, for example, under theconditions as follows: the concentration of the FAOD-αS in the reactionsolution in the range from 10 to 30,000 U/l, the concentration of theblood cells in the reaction solution in the range from 0.1 to 5 vol %,the reaction temperature in the range from 20° C. to 50° C., thereaction period in the range from 1 minute to 1 hour, and the pH in therange from 6 to 9.

[0058] Next, the hydrogen peroxide formed by the FAOD-αS treatment ismeasured by causing a further redox reaction using a POD and acolor-developing substrate.

[0059] As the color-developing substrate,N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt, orthophenylenediamine (OPD), a substrate in which aTrinder's reagent and 4-aminoantipyrine are combined, and the like canbe used, for example. Examples of the Trinder's reagent include phenols,phenol derivatives, aniline derivatives, naphthols, naphtholderivatives, naphthylamine, and naphthylamine derivatives. Furthermore,in place of the aminoantipyrine, it is possible to use aminoantipyrinederivatives, vanillin diamine sulfonic acid, methylbenzothiazolinonehydrazone (MBTH), sulfonated methylbenzothiazolinone hydrazone (SMBTH),and the like. Among these color-developing substrates,N-(carboxymethylaminocarbonyl)-4,4′-bis (dimethylamino)diphenylaminesodium salt is particularly preferable.

[0060] The redox reaction usually is carried out in a buffer. Theconditions of the reaction are determined as appropriate depending onthe concentration of the hydrogen peroxide formed, etc. The conditionsare usually as follows: the concentration of the POD in the reactionsolution in the range from 10 to 100,000 IU/l; the concentration of thecolor-developing substrate in the reaction solution in the range from0.005 to 30 mmol/l; the reaction temperature in the range from 15° C. to37° C.; the reaction period in the range from 0.1 to 30 minutes; and thepH in the range from 5 to 9. Moreover, the type of the buffer is notparticularly limited, and for example, the same buffers as in theprotease treatment and the FAOD treatments can be used.

[0061] In the redox reaction, for example, when the color-developingsubstrate is used, the amount of the hydrogen peroxide can be determinedby measuring the degree of the color developed (i.e. absorbance) in thereaction solution with a spectrophotometer. Then, the amount of theglycated protein in the sample can be determined using the concentrationof the hydrogen peroxide and a calibration curve or the like, forexample. In the present embodiment, the amount of the glycated proteinis determined based on the amount of a glycated amino group in a sidechain of an amino acid residue.

[0062] The hydrogen peroxide formed by the degradation FAOD-α addedfirst reacts with catalase present in the blood sample (hemolyzedsample) and is removed. Thus, it does not have any influence on themeasurement of the hydrogen peroxide derived from the analyte formed bythe FAOD-αS. The hydrogen peroxide formed by the FAOD-α may be removedby adding catalase. When the hydrogen peroxide is removed by thereaction with catalase, in order to prevent the hydrogen peroxide formedby the FAOD-αS treatment to be performed later from also being removed,it is preferable to add excessive amounts of POD and color-developingsubstrate when adding the FAOD-αS. In this case, the POD preferably isadded so that its activity (U) becomes 5 to 100 times that of thecatalase added, for example.

[0063] The amount of the hydrogen peroxide can be determined not only bythe above-described enzymatic method using the POD etc. but also by anelectrical method, for example.

[0064] In this measurement, the protease treatment in not necessarilyperformed before the degradation FAOD-α treatment as described above,and may be performed after the FAOD-α treatment, for example. Asdescribed above, the protease treatment is carried out so that the FAODscan act more easily. However, since the FAOD-α treatment is carried outin order to degrade the glycated amino acid, the effect of the presentinvention can be obtained sufficiently even if the glycated protein isnot degraded with the protease prior to the FAOD-α treatment.

[0065] The method for measurement according to the present embodimentalso is applicable, for example, when the non-analyte glycation productis a glycated amino acid having a glycated α-amino group and theglycated amine as the analyte is a glycated protein having a glycatedα-amino group. In this case, even if a degradation FAOD does not act onthe glycated protein easily, there is a possibility that the remainingdegradation FAOD may act on the glycated protein degradation productobtained by the protease treatment, for example. On this account, it ispreferable to inactivate the remaining FAOD by the degradation treatmentwith a protease as in a second embodiment described later, or to adjustthe ratio of a degradation FAOD to a measurement FAOD added to thesample as in a third embodiment described later.

[0066] (Second Embodiment)

[0067] The present embodiment is one example of the second method inwhich the same FAOD is used to degrade a glycated amino acid as anon-analyte glycation product and to measure a glycated protein as ananalyte. The FAOD used in not particularly limited, and for example, anyof a FAOD-α, a FAOD-S, and a FAOD-αS may be used.

[0068] A hemolyzed sample is prepared in the same manner as in the firstembodiment, and a degradation FAOD is added to this hemolyzed sample.

[0069] The treatment is carried out, for example, under the conditionsas follows: the concentration of the FAOD in the reaction solution inthe range from 10 to 5000 U/l, the concentration of the blood cells inthe reaction solution in the range from 0.5 to 20 vol %, the reactiontemperature in the range from 20° C. to 50° C., the reaction period inthe range from 1 minute to 1 hour, and the pH in the range from 6 to 9.This treatment usually is carried out in a buffer, and the same buffersas described above also can be used in this treatment.

[0070] Next, the sample treated with the FAOD is treated with aprotease. A first object of this protease treatment is to degrade theglycated protein derived from blood cells so that a measurement FAOD tobe added later can act thereon more easily, as described above. A secondobject of the protease treatment is to inactivate the degradation FAODby digesting it.

[0071] Since FAODs have properties that they do not act on glycatedproteins easily whereas they act on glycated amino acids easily, theglycated amino acid in the sample is degraded first in the treatmentwith the degradation FAOD. However, if the glycated protein is treatedwith the protease in the state where the degradation FAOD still remains,there arises a problem in that the remaining FAOD reacts with theglycation site of the glycated protein degradation product so that theglycated protein cannot be measured accurately. This problem can besolved by inactivating the remaining FAOD with the protease to preventthe remaining FAOD from reacting with the glycated protein degradationproduct. To this end, the amount of the protease to be added needs to besufficient to allow the degradation FAOD added first to be inactivatedrapidly and also the glycated protein to be degraded.

[0072] The type of the protease is not particularly limited, and thesame proteases as described above also can be used. The conditions ofthe protease treatment are determined as appropriate depending on thetype of the protease used, the type and the concentration of theglycated protein, the type and the amount of the degradation FAOD, etc.

[0073] The protease is added so that its concentration in the reactionsolution of the protease treatment falls, for example, in the range from1 to 1,000,000 KU/l, preferably from 10 to 300,000 KU/l, and morepreferably from 100 to 100,000 KU/l, when the concentration of thedegradation FAOD is 100 U/l.

[0074] Specifically, when the sample is treated using trypsin as theprotease, the protease treatment is carried out, for example, under theconditions as follows: the concentration of the protease in the reactionsolution in the range from 1000 to 30,000 KU/l; the concentration ofblood cells in the reaction solution in the range from 0.2 to 5 vol %;the concentration of the FAOD in the reaction solution in the range from10 to 1000 U/l; the reaction temperature in the range from 20° C. to 50°C.; the reaction period in the range from 10 minutes to 20 hours; andthe pH in the range from 6 to 9.

[0075] Subsequently, the same FAOD as the degradation FAOD is addedagain as a measurement FAOD to treat the glycated protein degradationproduct obtained by the protease treatment. It is necessary to add asufficient amount of the measurement FAOD because there is a possibilitythat the measurement FAOD may be inactivated with the protease.

[0076] The measurement FAOD treatment also preferably is carried out ina buffer as in the above. The type of the buffer is not particularlylimited, and the same buffers as in the protease treatment also can beused in this measurement FAOD treatment.

[0077] The measurement FAOD is added so that its concentration in thereaction solution of this measurement FAOD treatment is, for example, inthe range from 10 to 1,000,000 U/l, preferably 100 to 200,000 U/l, andmore preferably 500 to 50,000 U/l when the concentration of the proteaseis 10,000 KU/1.

[0078] Specifically, the conditions of the measurement FAOD treatmentare, for example, as follows: the concentration of the FAOD in thereaction solution in the range from 500 to 20,000 U/l; the concentrationof the protease in the reaction solution in the range from 100 to 30,000KU/l; the concentration of blood cells in the reaction solution in therange from 0.01 to 1 vol %; the reaction temperature in the range from15° C. to 40° C.; the reaction period in the range from 1 minute to 1hour; and the pH in the range from 6 to 9.

[0079] According to the present embodiment, even if the same FAOD isused to degrade the glycated amino acid and to measure the glycatedprotein, the glycated protein can be measured with high accuracy withoutbeing affected by the glycated amino acid.

[0080] (Third Embodiment)

[0081] The present embodiment is an example where the same FAOD is usedto degrade a glycated amino acid as a non-analyte glycation product andto measure a glycated protein as an analyte. However, the presentembodiment differs from the above-described second embodiment in that itis not always necessary to inactivate a degradation FAOD with aprotease. Because of the substrate specificity of enzymes, inactivatinga FAOD with a protease can be difficult depending on the combination ofthe FAOD and protease. A method for measurement according to the presentembodiment is effective in such a case. If a degradation FAOD addedfirst reacts with a glycated protein degradation product formed by thetreatment with a protease, the accuracy of the measurement cannot beimproved. Accordingly, it is important to adjust the ratio of adegradation FAOD to a measurement FAOD added to a sample as describedlater.

[0082] First, a hemolyzed sample is prepared in the same manner as inthe first embodiment, and a degradation FAOD is added to this hemolyzedsample.

[0083] When it is difficult to inactivate the degradation FAOD with theprotease used, the degradation FAOD needs to be added in an amount suchthat, even if the activity of the degradation FAOD remains during theprotease treatment, it does not act on the glycated protein degradationproduct formed. FAODs have properties that they do not act on glycatedproteins easily and act on glycated amino acids still more easily thanon glycated peptides. Therefore, the amount of the degradation FAOD tobe added and the reaction period preferably are set so as to allow thedegradation FAOD to act only on the glycated amino acid, for example.

[0084] The conditions of the FAOD treatment are, for example, asfollows: the concentration of the FAOD in the reaction solution in therange from 10 to 5000 U/l; the concentration of blood cells in thereaction solution in the range from 0.2 to 20 vol %; the reactiontemperature in the range from 20° C. to 50° C.; the reaction period inthe range from 1 minute to 1 hour; and the pH in the range from 6 to 9.This treatment usually is carried out in a buffer, and the same buffersas described above also can be used in this treatment.

[0085] Next, the sample treated with the FAOD is treated with aprotease. Since the present embodiment is an example where the proteasehardly acts on the FAOD, the amount of the protease to be added is notparticularly limited.

[0086] The type of the protease is not particularly limited, and thesame proteases as described above also can be used. The conditions ofthe protease treatment are determined as appropriate depending on thetype of the protease used, the type and the concentration of theglycated protein as the analyte, the type and the concentration of theFAOD added first, and the substrate specificity of the protease usedwith respect to the FAOD, etc., as described above.

[0087] Examples of the combination of a FAOD and a protease fallingwithin the present embodiment include the combination of a product namedFOD (Asahi Chemical Industry Co., Ltd.) and a product named Toyoteam(Toyobo Co., Ltd.) and the combination of a FAOD derived from the genusGibberella and a product named Proteinase K (Roche).

[0088] When the sample is treated using trypsin as the protease, theprotease treatment is carried out, for example, under the conditions asfollows: the concentration of the protease in the reaction solution inthe range from 100 to 6000 U/l; the concentration of blood cells in thereaction solution in the range from 0.2 to 5 vol %; the concentration ofthe FAOD in the reaction solution in the range from 0.1 to 100 U/l; thereaction temperature in the range from 20° C. to 50° C.; the reactionperiod in the range from 10 minutes to 20 hours; and the pH in the rangefrom 6 to 9.

[0089] Subsequently, the same FAOD as the degradation FAOD is addedagain as a measurement FAOD so that it acts on the glycated proteindegradation product obtained by the protease treatment.

[0090] The measurement FAOD treatment also preferably is carried out ina buffer as in the above. The type of the buffer is not particularlylimited, and the same buffers as in the protease treatment also can beused in this measurement FAOD treatment.

[0091] Thus, in the present embodiment, the ratio (activity ratio A: B)of the degradation FAOD (A) to the measurement FAOD (B) added to thesample is set, for example, in the range from 1:50,000 to 1:10,preferably 1:5000 to 1:25, and more preferably 1:500 to 1:50, asdescribed above. Unlike the above-described second embodiment, thedegradation FAOD remains in the reaction solution in the presentembodiment. However, when the ratio is in the above-described range, theremaining degradation FAOD does not act on the glycated proteindegradation product during the protease treatment to such an extent thatit affects the measurement because the reaction velocity of theremaining degradation FAOD is very low.

[0092] The conditions of the measurement FAOD treatment are, forexample, as follows: the concentration of the FAOD in the reactionsolution in the range from 500 to 20,000 U/l; the concentration of theprotease in the reaction solution in the range from 100 to 30,000 KU/l;the concentration of blood cells in the reaction solution in the rangefrom 0.01 to 1 vol %; the reaction temperature in the range from 15° C.to 40° C.; the reaction period in the range from 1 minute to 1 hour; andthe pH in the range from 6 to 9.

EXAMPLES Example 1 and Comparative Example 1

[0093] A fluid containing an amino acid and D-glucose was administeredto a patient via an intravenous drip, and the blood of the patient wascollected 1 hour later. The blood was centrifuged (1000 g, 10 min) toseparate blood cells and plasma. Then, 0.45 ml of the followinghemolysis reagent A was mixed with 0.006 ml of the blood cell fractionand 0.006 ml of the plasma fraction to hemolyze the blood cells. In thismanner, a plurality of hemolyzed samples were prepared. (HemolysisReagent A: pH 8.5) Product named TAPS (Dojindo Laboratories) 140 mmol/lGlycinamide (Nacalai Tesque, Inc.)  60 mmol/l Polyoxyethylene laurylether (Nacalai Tesque, Inc.)  24 g/l

[0094] Then, 0.0023 ml of solutions containing the following variousFAODs (concentration: 200 KU/l) respectively were added to the hemolyzedsamples at 25° C., and the resultant mixtures were incubated at 37° C.for 40 minutes. In the following paragraph, the (1) FAOD derived fromthe genus Penicillium is specific for a glycated α-amino group, the (2)FAOD derived from the genus Aspergillus is specific for a glycatedα-amino group and a glycated ε-amino group, and the (3) FAOX-E isspecific for a glycated α-amino group.

[0095] (Used FAOD)

[0096] (1) FAOD derived from the genus Penicillium (JP 8(1996)-336386 A)

[0097] (2) FAOD derived from the genus Aspergillus (WO 99/20039)

[0098] (3) Product named FAOX-E (Kikkoman Corporation, hereinafter thesame)

[0099] Next, to 0.01 ml of the hemolyzed samples respectively containingthe above-described FAODs were added 0.01 ml of purified water andfurther 0.065 ml of the following protease reagent, and the resultantmixtures were incubated at 37° C. for 5 minutes. Subsequently, 0.045 mlof the following color-developing reagent further was added, and theresultant mixtures were incubated at 37° C. for 3 minutes. Then, theabsorbance (at the wavelength of 751 nm) was measured with a measuringapparatus (product name JCA-BM 8, manufactured by Japan Electron OpticsLaboratory Co. Ltd.). On the other hand, as Comparative Example 1, themeasurement was carried out in the same manner as in Example 1 exceptthat purified water was added to a hemolyzed sample instead of thevarious FAODs. Furthermore, as a control test, the measurement wascarried out in the same manner as in Example 1 except that purifiedwater was mixed with blood cells instead of the plasma. The results areshown in Table 1 below. (Protease Reagent: pH 6.5) MOPS (DojindoLaboratories) 5 mmol/l Tetrazolium compound 2 mmol/l (product nameWST-3, Dojindo Laboratories) NaN₃ (Nacalai Tesque, Inc.) 0.05 g/l CaCl₂(Nacalai Tesque, Inc.) 5 mmol/l NaCl (Nacalai Tesque, Inc.) 300 mmol/lMetalloproteinase 3 g/l (Color-Developing Reagent) FAOD derived from thegenus Gibberella 26.0 KU/l (JP 8(1996)-154672 A) POD (Toyobo Co., Ltd)77.6 KU/l Color-developing substrate 0.052 mmol/l (product name DA-64,Wako Pure Chemical Industries, Ltd.) Tris-HCl buffer (pH 6.9) 200 mmol/l

[0100] TABLE 1 Absorbance after Type of FAOD 40 minutes (Abs.) Controltest FAOD: added/Plasma: not added 0.008 Ex. 1 (1) FAOD derived from thegenus 0.009 Penicillium (2) FAOD derived from the genus 0.009Aspergillus (3) Product named FAOX-E 0.008 Com. Ex. 1 FAOD: not added0.020

[0101] As shown in Table 1, since the glycated amino acid contained inthe plasma also reacted with the FAOD contained in the color-developingreagent in Comparative Example 1, the higher absorbance was exhibited inComparative Example 1 than in the control test by which only theglycated protein contained in the blood cells was measured. In contrast,the glycated protein could be measured accurately in Example 1 becausethe glycated amino acid contained in the plasma was treated with theFAOD in advance, and hence, Example 1 exhibited a high correlation withthe control test. This is because the FAOD contained in thecolor-developing reagent could act only on the degradation product ofthe glycated protein derived from the blood cells in Example 1.

Example 2 and Comparative Example 2

[0102] The blood of the patient after who had been put on an intravenousdrip was collected in the same manner as in Example 1 and was left tostand still. Then, the blood cells having precipitated naturally werecollected, and 0.01 ml of this blood cell fraction was mixed with 0.3 mlof the following hemolysis reagent B to prepare a hemolyzed sample. TheHb concentration and HbA1c concentration of this hemolyzed sample wereanalyzed with the above-described measuring apparatus (automaticanalysis apparatus). Since the blood cells having precipitated naturallywere collected, the blood cell fraction contained components in plasma.(Hemolysis Reagent B: pH 8.5) Product named TAPS (Dojindo Laboratories)140 mmol/l Glycinamide (Nacalai Tesque, Inc.)  60 mmol/l Polyoxyethylenelauryl ether (Nacalai Tesque, Inc.)  24 g/l Product named FAOX-E  1 KU/l(Kikkoman Corporation, hereinafter the same)

[0103] To 0.01 ml of the hemolyzed sample were added 0.01 ml of purifiedwater and 0.065 ml of the above-described protease reagent, and theresultant mixture was incubated at 37° C. The absorbance at thewavelength of 571 nm was measured after 4.5 minutes from the start ofthe incubation. The absorbance thus measured was regarded as theabsorbance showing the Hb concentration. Then, after 5 minutes from thestart of the incubation, 0.045 ml of the same color-developing reagentas used in Example 1 was added, and the resultant mixture was incubatedat 37° C. for 3 minutes. The mixture was allowed to react further. Then,after 3 minutes from the start of the reaction, the absorbance at thewavelength of 751 nm was measured using the above-described automaticanalysis apparatus. The absorbance thus measured was regarded as theabsorbance showing the HbA1c concentration.

[0104] Thereafter, the thus-measured absorbances were substituted intopreviously prepared calibration curves showing the relationships betweena Hb concentration (g/l) and absorbance and between a HbA1cconcentration (g/l) and absorbance, respectively, to determine the Hbconcentration and the HbA1c concentration. Then, HbA1c % was calculatedusing the following equation.

HbA1c (%)=(HbA1c concentration/Hb concentration)×100

[0105] The calibration curves were prepared in the following manner.First, standard solutions with various known concentrations of HbA1c andHb were provided. Then, the HbA1c concentration and the Hb concentrationof these standard solutions were measured using an automatic measuringapparatus (product name HA-8150, manufactured by ARKRAY, INC.). On theother hand, with respect to these standard solutions, the absorbancecorresponding to the HbA1c concentration and the absorbancecorresponding to the Hb concentration were measured in the same manneras described above. Based on the measured values given by the automaticmeasuring apparatus and the absorbances thus measured, primaryregression equations were prepared, which were used as the calibrationcurves.

[0106] On the other hand, as Comparative Example 2, the measurement wascarried out in the same manner as in Example 2 except that the hemolysisreagent A not containing the product named FAOX-E was added to the bloodcells instead of the hemolysis reagent B.

[0107] Furthermore, as a control test, the measurement was carried inthe following manner. To 0.05 ml of the blood cell fraction collectedafter letting blood cells precipitate naturally was added 2.5 ml of adiluent dedicated for the automatic measuring apparatus HA-8150 to causehemolysis, thus preparing a hemolyzed sample. The HbA1c concentration(%) of this hemolyzed sample was measured with the automatic measuringapparatus (the product name HA-8150: available from ARKRAY, INC.).

[0108] The results of the above-described measurements are shown inFIG. 1. FIG. 1 is a graph showing the relationship between the HbA1c (%)in Example 2 and Comparative Example 2 measured by the enzymatic methodand the HbA1c (%) obtained by the automatic analysis as the controltest. In FIG. 1, Example 2 has a correlation equation of“y=1.079x−0.409” and a correlation coefficient of “0.967” whileComparative Example 2 has a correlation equation of “y=1.124x−0.559” anda correlation coefficient of “0.931”.

[0109] In Example 2, the exogenous glycated amino acid contained in theplasma was degraded by the FAOD (contained in the hemolysis reagent B)treatment carried out first, and the hydrogen peroxide formed by thistreatment was removed by the reaction with catalase present in thesample. Therefore, in the redox reaction caused by the FAOD added later,only hydrogen peroxide derived from the glycated protein in the bloodcells was formed. Thus, as shown in FIG. 1, the value obtained inExample 2 was very close to the value obtained by the automatic analysiswith respect to the sample containing no plasma as the control test.Furthermore, Example 2 exhibited the extremely high correlationcoefficient (0.967) with the control test. On the other hand, inComparative Example 2, the FAOD caused to act on the glycated proteinalso reacted with the glycated amino acid contained in the plasma. As aresult, more hydrogen peroxide was formed than was derived from theglycated protein. Accordingly, HbA1c (%) obtained in Comparative Example2 was greater than the HbA1c (%) obtained by the control test, andComparative Example 2 exhibited a lower correlation coefficient (0.931)with the control test than Example 2.

INDUSTRIAL APPLICABILITY

[0110] As specifically described above, according to the method ofpretreating a sample of the present invention, a glycated peptide or aglycated amino acid as a non-analyte glycation product contained in thesample can be degraded so as to be removed. Therefore, by carrying outmeasurement of a glycated amine with respect to the sample pretreated bythis method, the influence of the non-analyte glycation product can beeliminated, which allows excellent accuracy of the measurement to beachieved. Thus, when the sample is blood collected from a patient afterbeing put on an intravenous drip and thus contains an exogenous glycatedamino acid and the like that are present only temporarily, the influenceof these substances can be eliminated. Accordingly, by applying themethod to, for example, the measurement of a glycated hemoglobincontained in erythrocytes, the measurement can be carried out withhigher accuracy than in conventional methods, which further increasesthe importance of the glycated hemoglobin as an index in the diagnosisand the like of diabetes.

1. A method of pretreating a sample containing a glycated amine as ananalyte, comprising: causing a fructosyl amino acid oxidase to act on aglycated amino acid or a glycated peptide present in the sample otherthan the glycated amine as the analyte so as to remove the glycatedamino acid or the glycated peptide by degrading it.
 2. A method ofmeasuring an amount of a glycated amine as an analyte in a sample,comprising: pretreating the sample by the method according to claim 1 soas to remove the glycated amino acid or the glycated peptide present inthe sample other than the glycated amine as the analyte by degrading it;then causing a fructosyl amino acid oxidase to act on the glycated amineto cause a redox reaction; and measuring the redox reaction to determinethe amount of the glycated amine.
 3. The method according to claim 2,wherein the fructosyl amino acid oxidase caused to act on the glycatedamino acid or the glycated peptide has a substrate specificity differentfrom that of the fructosyl amino acid oxidase caused to act on theglycated amine.
 4. The method according to claim 3, wherein thefructosyl amino acid oxidase caused to act on the glycated amino acid orthe glycated peptide is specific for a glycated α-amino group, and thefructosyl amino acid oxidase caused to act on the glycated amine isspecific for a glycated α-amino group and a glycated side chain of anamino acid residue.
 5. The method according to claim 3, furthercomprising: degrading the glycated amine with a protease to give adegradation product of the glycated amine either before or after causingthe fructosyl amino acid oxidase to act on the glycated amino acid orthe glycated peptide, wherein the fructosyl amino acid oxidase caused toact on the glycated amine is caused to act on the degradation product tocause the redox reaction.
 6. The method according to claim 4, whereinthe glycated amino acid present in the sample other than the glycatedamine as the analyte is a glycated amino acid having a glycated α-aminogroup, and the glycated amine as the analyte is a glycated protein or aglycated peptide having a glycated α-amino group and a glycated sidechain of an amino acid residue.
 7. The method according to claim 2,further comprising: degrading the glycated amine with a protease to givea degradation product of the glycated amine after causing the fructosylamino acid oxidase to act on the glycated amino acid or the glycatedpeptide, wherein the redox reaction is caused by adding the samefructosyl amino acid oxidase as that caused to act on the glycated aminoacid or the glycated peptide so that it acts on the degradation product.8. The method according to claim 7, wherein the fructosyl amino acidoxidase caused to act on the glycated amino acid or the glycated peptideis inactivated with the protease.
 9. The method according to claim 7,wherein the fructosyl amino acid oxidase (A) caused to act on theglycated amino acid or the glycated peptide and the fructosyl amino acidoxidase (B) caused to act on the glycated amine are added to the sampleso that a ratio (activity ratio A:B) of the fructosyl amino acid oxidase(A) to the fructosyl amino acid oxidase (B) is in a range from 1:10 to1:50,000.
 10. The method according to claim 7, wherein a glycation siteof the glycated amino acid or a glycated peptide is an α-amino group,the glycated amine as the analyte is a glycated protein, and a glycationsite of the glycated amine is an α-amino group.
 11. The method accordingto claim 2, wherein the glycated amine is at least one substanceselected from the group consisting of glycated amino acids, glycatedpeptides, and glycated proteins.
 12. The method according to claim 11,wherein the glycated proteins are glycated hemoglobins.
 13. The methodaccording to claim 5, wherein the protease is at least one proteaseselected from the group consisting of metalloproteinases, bromelain,papain, trypsin, proteinase K, subtilisin, and aminopeptidase.
 14. Themethod according to claim 13, wherein the protease is at least oneprotease that degrades a glycated hemoglobin selectively and is selectedfrom the group consisting of metalloproteinases, bromelain, papain,trypsin derived from porcine pancreas, and protease derived fromBacillus subtilis.
 15. The method according to claim 2, wherein thesample is at least one biological sample selected from the groupconsisting of whole blood, plasma, serum, blood cells, urine, and spinalfluid.
 16. The method according to claim 15, wherein the sample is awhole blood sample collected from a patient after being put on anintravenous drip.
 17. The method according to claim 2, wherein theglycated amino acid present in the sample other than the glycated amineas the analyte includes an exogenous glycated amino acid.